• Introduction

• Oligonucleotide Design for DNA Microarrays: A Thermodynamics Approach

• Pangenomic Database of Oligonucleotides

• Fabrication

• In Situ  Synthesis

• Applications

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• Gene Synthesis

• Application: Synthesis of the CYB5 Gene

Gene Synthesis

     There is substantial interest in implementing a bioinformatics tool that allows the design of oligonucleotides to support the development of in vitro gene synthesis. Current protocols to make long synthetic DNA molecules rely on the in vitro assembly of a set of short oligonucleotides, either by ligase chain reaction (LCR) or by assembly PCR. Ideally, such oligonucleotides should represent both strands of the final DNA molecule. They should be adjacent on the same strand and overlap the complementary oligonucleotides from the second strand to ensure good hybridization during assembly. This implies that the thermodynamic properties of each oligonucleotide have to be consistent across the set. Furthermore, any given oligonucleotide has to be totally specific to its target to avoid the creation of incorrectly assembled sequences. We have developed Gene2Oligo, a web-based tool that divides a long input DNA sequence into a set of adjacent oligonucleotides representing both DNA strands. The length of the oligonucleotides is dynamically optimized to ensure both the specificity and the uniform melting temperatures necessary for in vitro gene synthesis. We have successfully designed and used a set of oligonucleotides to synthesize the Saccharomyces cerevisiae cytochrome b5 by using both LCR and assembly PCR.

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Application: Synthesis of the CYB5 Gene

     We have processed the CYB5 sequence with Gene2Oligo using a hybridization unit size of 20 nucleotides and the software optimized T_m option. The sodium and DNA concentrations were left unchanged. We have obtained a total of 21 oligonucleotides with hybridization unit sizes ranging from 16 to 22 oligonucleotides and T_m ranging from 62.5 to 69.6°C.

     These oligonucleotides have been assembled into a full sized gene using either LCR or assembly PCR, followed by a PCR amplification. Here are the results:

(more detail)

Analysis of the synthetic CYB5 gene. 10 ul of each sample were analyzed on a 1% agarose gel. Lanes: L) 100 bp DNA ladder (NEB); 1) negative control, second PCR without template DNA; 2) LCR product before second PCR; 3) second PCR without primer after LCR; 4) second PCR after LCR; 5) assembly PCR product before second PCR; 6) second PCR without primer after assembly PCR; 7) second PCR after assembly PCR; 8) positive control, second PCR using yeast genomic DNA as template. The arrow indicates the 388 bp CYB5 PCR product.
 

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Last Updated: December 2004

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